Sponsoring Institution
National Institute of Food and Agriculture
Project Status
Funding Source
Reporting Frequency
Accession No.
Grant No.
Project No.
Proposal No.
Multistate No.
Program Code
Project Start Date
Sep 1, 2013
Project End Date
Aug 31, 2015
Grant Year
Project Director
Bennett, G. M.
Recipient Organization
AUSTIN,TX 78712-1500
Performing Department
Section of Integrative Biology
Non Technical Summary
Leafhoppers (Hemiptera: Cicadellidae) are one of the largest insect families, containing many of the most notorious invasive pest species in North America. While most leafhoppers remain ecologically restricted, others have emerged as devastating pests that are able to feed hundreds of plant species and to persist in a wide range of environments. The ability of leafhoppers to emerge as widespread pests requires adaptive environmental and dietary flexibility that is potentially limited by relationships with bacteria. Many bacteria that live in insects are dependent on their hosts, and the hosts on them; and, these relationships have persisted for millions of years. Bacterial symbionts provide their hosts with essential nutrition that is absent in the specialized diets of leafhoppers - plant saps that lack many nutrients animals require such as amino acids. In turn, the host must regulate symbiont numbers, function, and metabolic contributions since symbionts have tiny, degraded genomes that lack many of the genes required for maintaining independent bacterial life. Insect-bacteria relationships further confer many other adaptive capabilities regarding some environmental challenges, while imposing significant limitations regarding others (e.g., thermal tolerance, host plant choice host plant choice, and parasitoid resistance). Thus, the coordination of symbioses must entail complex systems that present major challenges when hosts encounter changing environmental conditions and food-plant availability. This leaves the important questions of what role symbioses play in the adaptation and the emergence of pest species? And, how are symbioses functionally regulated, especially in response to changing environmental and dietary conditions? Our project will address how insect-bacteria symbioses are functionally integrated in a pest insect, and how this system responds to changing climate and dietary conditions. We will use the pest species, Macrosteles quadrilineatus, as a model to understand the major tasks of symbiosis. Macrosteles quadrilineatus relies on two obligate symbionts for nutrition and other metabolic needs. We propose a comprehensive experimental approach that will elucidate the mechanisms that regulate this symbiosis, and the role of the each symbiotic partner. Specifically, we will use cutting-edge genomic approaches to identify expressed genes that are from both the symbionts and the host insect, and the roles the regulatory roles these genes play. Finally, results from this study will elucidate the role of food-plant availability and changing environmental conditions in the persistence of local pest species and the potential emergence of novel ones.
Animal Health Component
Research Effort Categories

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
Goals / Objectives
The long-term goal of this proposal is to understand how insect symbioses are regulated, and what role symbioses play in insect adaptation - specifically as it applies to the emergence of invasive pest species. We will use the pest leafhopper species, Macrosteles quadrilineatus (Hemiptera: Cicadellidae), as a model to understand the major tasks of symbiosis, applying novel molecular technologies and experimental manipulations to address the following supporting objectives: 1. Determine the metabolic contributions of the dual bacterial symbionts and host. 2. Distinguish the mechanisms that regulate symbiont function and numbers. 3. Determine how symbioses in pestiferous species are regulated and adapt under environmental and dietary change.
Project Methods
Symbiont Genome Sequencing: Symbiont genome sequences will be completed with Illumina MIseq 250 paired-end inserts. Genomes will be assembled with the following pipeline: i) first pass de novo assembly with VELVET with a subset of the reads, ii) endosymbiont read extraction with SOAP, iii) refined de novo assembly with MIRA, and iv) final consensus scaffold assembly with all reads in SOAP. Gaps will be closed with PCR. Genes and pathways will be predicted with Joint Genome Institute's IMG ER pipeline. Experimental manipulations of temperature and diet: We will heat-shock leafhopper cultures under varying temperatures as a proxy for changing environmental conditions. Populations will be subjected to 25 (control rearing temp), 32, 40, and 45 0C for 3 hours and 24 hours. This temperature range encompasses summer month averages and conditions known to delay insect development and reduce survival rates. The time intervals assess immediate and sustained responses. Experimental treatments that simulate food plant quality will be done by adding two EAA supplements synthesized by only one of the symbionts: arginine (Sulcia), methionine (BetaSymb), all EAAs, no food (starvation treatment), and control (no treatment). Increased EAA concentrations will be achieved by adding dissolved supplements to cut plants as done in previous projects. Adults will be fed on EAA spiked cuttings for 24 hours. Each treatment will include a parallel population for monitoring demographics and fecundity. Treatment samples will be sequenced their complete transcriptomes and proteomes in replicate to ensure reproducibility of results. Population sizes of each endosymbiont will be quantified with quantitative-PCR (qPCR) using methods established in the Moran Lab. We have designed qPCR primer sets for the groEL locus for both symbionts and the nuclear locus wingless for leafhopper host. Host and Symbiont Gene Expression: For transcriptomic and proteomic analyses, specimens from each treatment will be pooled (10-20 individuals) and dissected into bacteriocyte types and host tissues. We will use micro-dissection techniques to separate the distinct bacteriocyte lobes that separately house each symbiont. These will be pooled and screened with PCR for the occurrence of one or more bacterial symbionts. Contaminated samples will be discarded. Verified tissue pools will be sequenced for the complete RNA transcriptome with paired-end Illumina HiSeq after reducing eukaryotic and prokaryotic ribosomal RNA. Sequence data will be assembled with TRINITY. Symbiont and host transcripts will be identified with genome templates from Obj. 1, and with existing insect genome databases. To manage costs, sequencing lanes will be split for multiple samples (10 per lane). This should give enough read coverage to successfully sequence the bacteriocyte transcriptomes (e.g., symbiont and host tissues). Transcript expression levels will be determined by mapping and normalization with RSEM and RPKM, and statistical differences in expression determined with EdgeR and DESeq. We will use quantitative proteomics to compare complete protein profiles of dissected tissue pools for each of the different treatments with iTRAQ (isobaric Tags for relative and absolute quantitation). Samples will be labeled with chemical tags, pooled, peptides separated with chromatography, and proteins identified with mass spectroscopy compared against known spectral profiles based on translated sequences. Treatments will exploit current technology that allows up to 8 samples to be processed in parallel. These protocols are in place at the Yale Keck Proteomics facility, or other services. Protein expression, quantitation, and statistical analyses will per performed with iTRAQPak.

Progress 09/01/13 to 08/31/15

Target Audience:The target audiences of this work include the scientific community and agricultural production and insect disease vector related researchers. This work was shared at several scientific conferences in the fields of Applied and Basic Entomology, Symbioses, and Molecular Evolution. This project was also shared with undergraduate students at the University of Texas Austin as experiential educational experience. These students participated in actual research projects related to the proposal. Finally, the results of this work have been shared with Agricultural scientist at the University of Hawaii, Manoa, in the College of Tropical Agriculture and Human Resources. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This fellowship provided the basis for a new research focus for Dr. Bennett (PI). This includes agricultural pest species, plant pathogen vectors, and microbial symbioses. Dr. Bennett recently started a tenure track faculty position at the University of Hawaii Manoa, in the College of Tropical Agriculture and Human Resources, Department of Plant and Environmental Protection Sciences. The opportunity provided by this fellowship for him to pursue research agriculturally related systems, directly contributed to his career trajectory and goals. It has also expanded Dr. Bennett' collaborative network through agricultural communities. His work will continue to focus on the microbial symbionts and pathogens in native and pestiferous agricultural species. The fellowship further provided collaborative opportunities for Dr. Bennett with European pest insect and pathogen research labs. He has developed active collaborations with Dr. Cristina Marzachi and Dr. Simona Abba at the Institute for Sustainable Plant Protection, National Research Council of Italy, Torino. During this collaboration, Dr. Bennett visited Dr. Marzachi's lab and conducted molecular genomic work, presented on his projects, and conducted fieldwork in local vineyards. This included work on microbial symbionts and phytoplasma pathogens. Dr. Bennett is continuing collaborations with Dr. Marzachi during his new position at UH. Dr. Bennett mentored two undergraduate researchers for two years under this project. Both participated in several project dimensions. They were trained in experimental setup, insect rearing and experimentation, and molecular bench work. Specifically, they worked to develop an artificial diet for insect experiments, ran insect feeding trials on different nutrient profiles, extracted and sequenced DNA, conducted dissections and microscopy work, and helped organize data from molecular and genomic experiments. They also presented their results to general scientific audiences. Both have gone on to pursue scientifically related jobs in human pathogens and medicinal sciences. How have the results been disseminated to communities of interest?Project results have been presented at multiple conferences and seminars, targeting entomological, phytopathological, and evolutionary scientific communities (see products for explicit list of contributions). All results have been actively published in relevant scientific journals (at total of six publication thus far). Much of this work is still on going, and will be published upon completed. Dr. Bennett, through his new position at UH Manoa's premier Agricultural College, is working with extension personnel throughout Hawaii to share information about microbial symbionts and pathogens in introduced pests. To date, he has made several trips to Hawaii Island to meet with local Forest Service employees, Hawaii Department of Agriculture workers, and Hawaii Department of Land and Natural Resources land managers. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

What was accomplished under these goals? Objective 1: Dr. Bennett sequenced the genomes of the two obligate symbionts (Nasuia deltocephalinicola and Sulcia muelleri) of the leafhopper pest, Macrosteles quadrilineatus (Cicadellidae). This revealed the smallest and degraded bacterial genomes known to science. Results show that symbionts are required for the synthesis of essential amino acids (EAA) that animals cannot make de novo and are limited in insect phloem-sap diets. Both symbionts require extensive cellular resources from their host for basic cellular and metabolic functions that include energy (e.g., ATP), cell membrane and transport, and cellular division. However, these requirements differ between symbionts as their genomes encode different functions and metabolisms. Bacterial symbionts further appear to require assistance in initiating the synthesis of EAAs (e.g., methionine) required by the host. Preliminary gene expression analyses with RNA-seq sequencing have demonstrated that Macrosteles quadrilineatus provides functions that are missing from symbiont genomes. RNA expression profiles for host symbiont organs (bacteriomes) and non-symbiotic insect tissues were comparatively interrogated. Results reveal that the host has increased expression in the bacteriomes of genes involved in the transport of amino acids and sugars, cell membrane components, cell division, and EAA synthesis. These outcomes reveal potential targets for future gene expression knockdown experiments to disrupt symbiotic functions (e.g., RNAi). Other pathogenic bacteria were identified in the genomic data for insect tissues. The insect pathogen Arsenophonus sp. appears is present in M. quadrilineatus tissues. While Arsenophonus can have life history and ecological effects on insect hosts, its role in M. quadrilineatus is currently unknown. Project results under Objective 1 were extended through collaboration with Dr. Cristina Marzachi and Dr. Simona Abba at the Institute for Sustainable Plant Protection, National Research Council of Italy, Torino. This collaboration profiled and assembled the genomes of the microbes associated with the European pest leafhopper, Macrosteles quadripunctulatus. Results demonstrate that M. quadripunctulatus harbors the same microbial symbionts required for nutritional synthesis as M. quadrilineatus. The symbiont genomes pf host species are nearly identical. Sequencing results further found additional microbes infecting M. quadripunctulatus that include "Candidatus Phytoplasma" plant pathogens and the insect pathogen, Rickettsia. Objective 2: Results for Objective 1 have revealed a suite of up-regulated genes for the maintenance and function of bacterial symbionts in M. quadrilineatus. However, these data do not differentiate which genes are targeted for the maintenance of either Sulcia or Nasuia. In order to distinguish the mechanisms that support individual symbiont types, Dr. Bennett developed a micro-dissection technique to split host cells (bacteriocytes) that contain Sulcia or Nasuia, which are sequestered separately. Dissected tissue pools will be interrogated for host and symbiont gene expression (RNAseq). To date, Dr. Bennett has completed dissections and sequencing for three technical replicate series (3 sets of 30 individuals). These pools were sequenced using RNA-seq. Preliminary results of host gene expression show widely different profiles for each symbiont specific cell type. This is because the Nasuia and Sulcia encode for drastically different functional and metabolic abilities and require different input from the host insect. Analysis of differential gene expression, gene identification, and metabolic networks are ongoing. Data collected under Objective 2 has also revealed bacterial gene expression differences between Nasuia and Sulcia. Results show that the most highly expressed bacterial genes are involved in stress repsonse and protein folding, as well as EAA metabolisms required by the hosts. The high expression of stress related proteins are hypothesized to result from the intracellular symbiont environment. Symbiont typically experience reduced natural selection and increased mutation rates that impair gene function. Thus, proteins are likely unable to fold and function properly. Stress response proteins are known to help aid in protein function. These results are being combined with a broader sequencing effort across other sap-feeding insects in the Hemiptera (e.g., psyllids and aphids) to determine if these expression patterns are widespread in symbiotic systems. Objective 3: Dr. Bennett established a lab-reared colony of M. quadrilineatus for environmental and dietary experiments. Two undergraduate research assistants helped develop the tools to conduct these experiments, Allison Joyce and Shivani Patel. To date, they have worked on developing delivery systems of dietary enrichments that include an artificial diet and spiking barley plant cuttings with EAA enrichment solutions. Tests have determined that plant cuttings work sufficiently and that artificial diets are unstable. Finally, they have developed quantitative PCR molecular markers for assaying symbiont population sizes. Dr. Bennett and his mentees completed experiments for age-controlled populations under different temperature and diet regimes: 10-20 individuals for across four different temperature profiles (15, 25, 30, 45 oC for 3 and 24 hours periods); and, four different EAA diet enrichments that are i) redundant with the role of Sulcia, ii) redundant with the role of Nasuia, and ii) complete EAA enrichment, and d) a control. They have extracted symbiont and host DNA and conducted quantitative PCR analyses. These analyses are used to determine fluctuations of symbiont populations in different conditions. Preliminary results show that the populations of Nasuia and Sulcia differ dramatically in abundance. Sulcia is over 3-fold more abundant than Nasuia. This may be due to Sulcia's overall larger contribution to the nutrient pool required by the host (i.e., 8 essential amino acids, as opposed to the 2 synthesized by Nasuia). Finally, females have larger symbiont populations than do males. Presumably this is because symbionts are passed to the next generation directly within the matriline. Insect diet appears to impact symbiont abundance. When available plant nutrients have increased amino acid concentrations redundant with the metabolisms of one of the symbionts, the other symbiont population size is adjusted. Preliminary results show that symbiont populations increase to balance the concentrations of all EAAs. That is, when we increased the EAA concentrations synthesized by Nasuia, Sulcia populations increased, but no difference is found in Nasuia populations. We hypothesize this is a compensatory response by the host to balance EAA profile, which can be toxic if unbalanced. Increasing symbiont numbers is one way to increase amino acid production, since it obligate symbionts generally cannot control gene expression. Project Extensions: In addition to NIFA fellowship project objectives, Dr. Bennett has also completed several side-projects that are extensions of this work. These include the genomic sequencing of bacterial symbionts in other pestiferous leafhopper species, including the blue-green sharpshooter, Graphocephala atropunctata (Bennett et al., 2014, mBio), and the green sharpshooter Draeculacephala minerva (Bennett et al., 2015, Genome Biology and Evolution). Dr. Bennett and Dr. Nancy Moran have also co-authored two perspective pieces on the evolution of obligate symbioses (Moran & Bennett 2014, Nat. Rev. Micrbiol.; and, Bennett & Moran 2015, Proc. Nat. Acad. Sci. USA).


  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Moran, N.A. and Bennett, G.M. 2014. The tiniest tiny genomes. Annual Review Microbiology. 68:195-215.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Bennett, G.M., McCutcheon, J.P., McDonald, B., Romanovich, D., & Moran, N.A. 2014. Differential genome evolution between companion symbionts in an insect-bacteria symbiosis. mBio. 5:e01697-14
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Bennett, G.M., McCutcheon, J.P., McDonald, B., Romanovich, D., & Moran, N.A. 2015. Lineage-specific patterns of genome deterioration in obligate symbionts of sharpshooter leafhoppers. Genome Biology and Evolution. [early edition]
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Bennett, G.M. and Moran, N.A. 2015. Heritable symbiosis: the advantages and perils of an evolutionary rabbit hole. Proceedings of the National Academy of Sciences, USA. 112:10169-10176
  • Type: Journal Articles Status: Accepted Year Published: 2015 Citation: Bennett, G.M., Abba, S., Kube, M., Marzachi, C. 2015. Complete genome sequences of the obligate symbionts "Candidatus Sulcia muelleri" and "Candidatus Nasuia deltocephalinicola from the pestiferous leafhopper Macrosteles quadripunctulatus (Hemiptera: Cicadellidae) [in press]
  • Type: Book Chapters Status: Awaiting Publication Year Published: 2016 Citation: Bennett, G.M. The microbial symbionts of leafhoppers. In: Webb, M., and Badmin, J. (Ed.) The Leafhoppers: Form Function and Phylogeny. [In press]